Advanced Functional Biocompatible Polymeric Matrix Used as a Hemostatic Agent and System for Damaged Tissues and Cells

ABSTRACT

A hemostatic tissue sealant sponge and a spray for acute wounds are disclosed. The sponge comprises hydrophobically modified polymers that anchor themselves within the membrane of cells in the vicinity of the wound. The seal is strong enough to substantially prevent the loss of blood inside the boundaries of the sponge, yet weak enough to substantially prevent damage to newly formed tissue upon recovery and subsequent removal of the sponge. In examples, the polymers inherently prevent microbial infections and are suitable for oxygen transfer required during normal wound metabolism. The spray comprises hydrophobically modified polymers that form solid gel networks with blood cells to create a physical clotting mechanism to prevent loss of blood. In an example, the spray further comprises at least one reagent that increases the mechanical integrity of the clot. In another example, the reagent prevents microbial infection of the wound.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/001,215 filed on Jan. 19, 2016; which is a continuation of U.S.patent application Ser. Nos. 14/595,542 and 14/595,551 filed on Jan. 13,2015; which are continuations of U.S. patent application Ser. No.12/231,571, filed on Sep. 4, 2008, now U.S. Pat. No. 8,932,560, whichclaims priority under 35 U.S.C. § 119 to the U.S. ProvisionalApplication Ser. No. 60/969,721, filed on Sep. 4, 2007; which are allherein incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention generally relates to the field of hydrophobicallymodified polymers and their use in promoting hemostasis in woundedtissues and cells.

BACKGROUND OF THE INVENTION

Currently, every year 21 million people worldwide suffer from seriousinjuries resulting in severe blood loss, and more than one third ofthese cases lead to death. (See Bledsoe, B. E.“The Golden Hour: Fact orFiction.” Emergency Med. Serv. 6, 105 (2002), which is hereinincorporated by reference in its entirety.) Among these patients areAmerican soldiers in Iraq whose fatality rate with severe injuries is90%. Uncontrolled hemorrhaging from these injuries is the leading causeof preventable combat deaths among U.S. soldiers in Iraq. According tothe Marine Corps registry, 45-60% of combat casualty deaths are due topotentially preventable uncontrolled hemorrhage. (See Clarke, PatrickE.“Z-Medica's Products Cited as Life Saving on Battlefield”, which isherein incorporated by reference in its entirety.) Similarly,uncontrolled hemorrhage is the leading cause of potentially preventabledeaths in the U.S with 30-40% of all trauma deaths in the civilianpopulation due to uncontrolled bleeding. In many cases, these deathsoccur before the injured are able to be transported to medical treatmentwith approximately 40% of civilian and 90% of military casualtiesoccurring before the patients reach a treatment facility. (See Kim,Seung-Ho M D; Stezoski, S. William; Safar, Peter M D; Capone, Antonio MD; Tisherman, Samuel M D. Journal of Trauma-Injury Infection & CriticalCare. 42(2):213-222, February 1997, which is herein incorporated byreference in its entirety.) Many of these casualties can be prevented ifan effective treatment is used to quickly stop the significant loss ofblood.

Controlling hemorrhage is also a critical issue in medical facilitieswith 97 million patients experiencing surgical bleeding every yearworldwide. (See B. S. Kheirabadi, E. M. Acheson, R. Deguzman, J. L.Sondeen, K. L. Ryan, A. Delgado, E. L. Dick, J. B. Holcomb, “Hemostaticefficacy of two advanced dressings in an aortic hemorrhage model inswine.” J. Trauma. (2005), which is herein incorporated by reference inits entirety.) Despite advances in medical technology, hemorrhagecontrol is still a major problem in emergency medical care. In the first48 hours of hospitalization, approximately 51% of deaths are due tohemorrhage (See F W Verheugt, M J van Eenige, J C Res, M L Simoons, P WSerruys, F Vermeer, D C van Hoogenhuyze, P J Remme, C de Zwaan, and FBaer. Bleeding complications of intracoronary fibrinolytic therapy inacute myocardial infarction. Assessment of risk in a randomised trial,which is herein incorporated by reference it its entirety.)

Improving the ability to control hemorrhage for injuries that areotherwise survivable would greatly reduce trauma mortality, and thisknowledge has encouraged numerous advancements in hemostatic control;however, the currently available hemostatic bandages are notsufficiently resistant to termination in high blood flow and they do nothave strong enough adhesive properties to stop severe blood flow for anadequate time period.

Today the application of continuous pressure using gauze bandagesremains the primary technique used to stanch blood flow, particularly insevere bleeding wounds. This procedure neither successfully nor safelystops severe blood flow. As in the past, this method continues to be amajor survival problem in the case of serious life-threatening bleeding.Other currently available hemostatic bandages, such as collagen wounddressings or dry fibrin thrombin wound dressings do not have strongenough adhesive properties to serve any realistic purpose in thestanching of severe blood flow. These hemostatic bandages are alsofragile and are therefore liable to fail if damaged due to bending orapplication of pressure. (See Gregory, Kenton W. and Simon J. McCarthy.Wound dressings, apparatus, and methods for controlling severe,life-threatening bleeding, which is herein incorporated by reference inits entirety.)

Recent advancement in hemostatic bandages have targeted the immediatetreatment of acute wounds, such as the prevention of casualties due tohemorrhage on the battlefield. Chitosan based bandages have beenapproved and used in numerous settings including battlefield use withsuccess. Chitosan is an amino-polysaccharide that is commerciallyproduced from the deacetlyation of chitin which is an abundant naturalbiopolymer that is found in the exoskeleton of crustaceans. Advantagesof chitosan as a material for wet wound dressings include its ability toaccelerate wound-healing, its hemostatic properties, its stimulation ofmacrophage activity, and its general anti-microbial impact which helpsprevent infection at the wound site. Chitosan is used as a hemostaticagent because of its cationic nature. Since the surfaces of mostbiological cells are anionic, including red blood cells, chitosanstrongly adheres to the cells of tissue at wound sites because of anelectrostatic interaction and is able to initially halt blood flow. (SeeDornard, Alain and Monique. “Chitosan: Structure-Properties Relationshipand Biomedical Applications.” (2002), which is herein incorporated byreference it its entirety.)

Despite the advantages of using a chitosan-based dressing, there arealso significant disadvantages. In a study conducted in 2005 byKheirabadi et al, researchers caused injury to the aorta of pigs andattempted to control hemorrhage using the Hemostatic HemConÂ® Bandage.The results of the study showed that though the chitosan bandageeffectively reached hemostasis immediately after its application,secondary bleeding resumed approximately 2 hours later and resulted inthe death of the pigs. They found that the adhesion between the bandageand the tissue decreased as the bandaged became saturated with blood.(See B. S. Kheirabadi, E. M. Acheson, R. Deguzman, J. L. Sondeen, K. L.Ryan, A. Delgado, E. L. Dick, J. B. Holcomb, “Hemostatic efficacy of twoadvanced dressings in an aortic hemorrhage model in swine.” J. Trauma.(2005), which is herein incorporated by reference in its entirety.) Inrealistic situations in which patients are incapable of reachingadequate medical treatment, such as on the battlefield, this time periodis too short and the Hemostatic HemConÂ® Bandage would be aninsufficient hemostatic agent.

Wound physiology is difficult to manipulate due to the complexity ofcell-to-cell signaling networks which communicate during wound healing,but fortunately for healthy individuals, severe wounds can be healedquite well by simply disinfecting the wound area and stopping the lossof blood by suturing the open, damaged tissue. However, in many cases ofacute wounds, internal or external, suturing is neither effective norpractical. In these cases, it is advantageous to use solid materialswhich adhere strongly enough to tissue such that they provide a sealupon pressing such materials onto the damaged tissue, thus preventingthe loss of blood within the boundaries of the seal.

Additionally, blood clotting is a necessary aspect of wound physiology,and in many practical cases it is highly valuable for survival. However,for human beings and most mammals, the ability for blood to clot islimited because a significant increase in the ability of healthymammalian organisms to form blood clots would most likely result indeath of the organism as a result of any minor internal injury. In suchcases, clots would form at the site of the injury, enter into the bloodstream and eventually cause a stroke or heart failure due to blockage ofimportant blood vessels. Because clotting ability must be limited undernormal physiological conditions, in the case of acute wounds, whichrefers to a fast impact injury resulting in a high rate of blood loss,clotting is not a legitimate means to provide hemostasis. Therefore,acute wounds typically drain blood banks of their stocks as large bloodtransfusions are required to keep patients alive during transport totreatment facilities and subsequently during the surgery required toclose the wound.

Therefore, it would be desirable to provide a strongly adhesivehemostatic bandage that promotes increased adhesion to wounded tissue orcells. It would also be desirable to provide a strongly adhesiveflowable spray solution or surgical sealant to stop minor bleeding andto seal tissues in surgical applications.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a novel hybrid compositionof matter that provides a strongly adhesive hemostatic agent. In apreferred exemplary embodiment, the composition of matter is a film orsolid state tissue sealant sponge that is composed of a hydrophobicallymodified biopolymer matrix capable of hydrophobically interacting withtissue, particularly hemostatic interaction with damaged tissue. It iscontemplated that this sponge may be included as part of a bandage orother applicator form as may be known by those skilled in the art. Thehemostatic interaction occurs through a process wherein a plurality ofshort hydrophobic substituent, that are attached with the polymerbackbone, interact with and adhere to the tissue.

In a further preferred exemplary embodiment, the composition of matteris formulated in a liquid state as a spray solution. The solution iscomposed, at least in part, of the hydrophobically modified biopolymermatrix capable of hydrophobically interacting with and/or gelling withcells, particularly a hemostatic interaction with tissue and/or redblood cells. Similar to the solid state film's interaction process, thehemostatic interaction of the solution occurs through a process whereina plurality of short hydrophobic substituent, that are attached with thepolymer backbone, interact with and adhere to the tissue and/or cells.

In other preferred exemplary embodiments, the current invention providesa system for delivering the novel solution including the functionalcapabilities of the hydrophobically modified polymer matrix. In oneexemplary embodiment, the system includes a container for storing thesolution and an ejection mechanism connected with the container to ejectthe solution to an environment outside the container. In an alternativeexemplary embodiment the system includes two or more containersoperationally connected through an ejection mechanism for ejecting amixture of the solution and various other secondary components.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention as claimed. The accompanyingdrawings, which are incorporated in and constitute a part of thespecification, illustrate an embodiment of the invention and togetherwith the general description, serve to explain the principles of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

The numerous advantages of the present invention may be betterunderstood by those skilled in the art by reference to the accompanyingfigures in which:

FIG. 1 is a structural and graphical illustration representing a readilyreactive, hydrophobically modified chitosan matrix(hm-Chitosan), whereinthe hm-Chitosan “backbone” or “scaffold” is capable of binding with aplurality of short hydrophobic substituents and the resulting compoundbeing formulated as a tissue sealant sponge (solid state) or solution(liquid state) in accordance with an exemplary embodiment of the presentinvention.

FIG. 2 is an illustration of a tissue sealant sponge showing the matrixincluding the plurality of short hydrophobic substituent that providethe hydrophobic interaction functional capability to the solid state andliquid state formulation of the current invention.

FIG. 3 is an illustration providing a representation of hydrophobicinteraction between the short hydrophobic substituents of the modifiedpolymer matrix and the bi-layer of tissue and/or cells.

FIG. 4 is a block diagram representation of a method of using thehydrophobically modified biopolymer matrix of the current invention.

FIG. 5 is a graph showing the Force required for removal of chitosan andhm-chitosan films vs. level of hydrophobic modification.

FIG. 6A is an illustration of 4-octadecyloxybenzaldehyde 2.5% modifiedhm-chitosan and blood solution when first mixed.

FIG. 6B is an illustration of -octadecyloxybenzaldehyde 2.5% modifiedhm-chitosan and blood solution immediately after mixture with invertedtest tube to show gelation of solution.

FIG. 7A is an illustration of a dynamic rheology frequency sweep showinga chitosan (0.75 wt %)+blood (heparinized).

FIG. 7B is an illustration of a dynamic rheology frequency sweep showing2.5% C12 mod hm-chitosan (0.75 wt %)+blood (heparinized).

FIG. 7C is an illustration of a dynamic rheology frequency sweep showing6% C12 mod hm-Chitosan (0.75 wt %)+blood (heparinized).

FIG. 7D is an illustration of a dynamic rheology frequency sweep showing2.5% mod (C18-benz) hm-Chitosan (0.4 wt %)+blood (heparinized).

FIG. 8A is an illustration of time to gelation showing a chitosan (0.75wt %)+blood (heparinized).

FIG. 8B is an illustration of time to gelation showing 2.5% C12 modhm-chitosan (0.75 wt %)+blood (heparinized).

FIG. 8C is an illustration of time to gelation showing 6% C12 modhm-Chitosan (0.75 wt %)+blood (heparinized).

FIG. 8D is an illustration of time to gelation showing 2.5% mod(C18-benz) hm-Chitosan (0.4 wt %)+blood (heparinized).

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to the presently preferredembodiments of the invention, examples of which are illustrated in theaccompanying drawings.

Referring generally to FIGS. 1 and 2, the current invention provides anovel composition of matter that is capable of promoting hemostasisand/or hemostatic response through hydrophobic interactions with tissueand/or cells. In preferred embodiments, the current invention provides ahydrophobically modified polymer matrix capable of hydrophobicinteractions with various tissue and/or cells to promote hemostasis. Thehydrophobically modified polymer provides a readily reactive matrix andis capable of maintaining its reactive nature through variousformulations, such as in a solid-state film or liquid state solution.

In a preferred embodiment, the current invention provides a solid state,hemostatic tissue sealant sponge that is composed of at least onepolymer and a plurality of short hydrophobic substituents attached alongthe backbone of the polymer is disclosed. The sponge extends thehemostatic lifespan of a polymer-based bandage due to anchoring of thehydrophobic grafts into the membranes of cells in the vicinity of thedamaged tissue. As a result, the sponge is an effective hemostaticsealant device. The level of hydrophobic modification of the polymer aswell as hydrophobic substituent type is substantially optimized todevelop materials which adhere to tissue in a manner idealized forclinical applications: the material comprising the sponge adheresstrongly enough to provide hemostasis for a long enough time period toallow for substantially full patient recovery, yet weakly enough suchthat newly formed tissue is substantially undamaged upon removal of thetissue sealant device after patient recovery.

In another preferred embodiment, a hemostatic spray solution (liquidstate) is composed of the hydrophobically modified polymer matrixproviding at least one water-soluble polymer and a plurality of shorthydrophobic substituent attached along the backbone of the polymer. Thehydrophobically modified polymer sprayed in aqueous solution is able toform solid networks upon interaction with blood, as the hydrophobicsubstituent are able to anchor themselves within the bilayers of bloodcell. The result is a localized “artificial clot” which physicallyprevents further blood loss around the newly formed solid network.“Artificial clots” herein refer to physical networks of hydrophobicallymodified polymers, blood cells, and surround tissue cells whicheffectively act as a solid barrier to prevent further blood loss.Additionally, the level of hydrophobic modification of the polymer aswell as hydrophobic substituent type can be substantially optimized toyield rapidly forming and mechanically robust artificial clots. In anexample, the hydrophobically modified polymer spray solution is mixedwith at least one water-soluble reagent that results in faster and moreefficient healing of the wound.

The novel hemostatic agent of the current invention is suitable for usewith any various tissues and cells. These agents may be used with thetissues and cells of mammals. As used herein, the term “mammals” meansany higher class of vertebrates that nourish their young with milksecreted by mammary glands, e.g. humans, rabbits and monkeys.

The polymer that forms the backbone of this reactive matrix is ofsynthetic or natural origin, including for example, water-solublepolysaccharides and water-soluble polypeptides. In particularlypreferred embodiments, the polymer is one or more hydrophobicallymodified polysaccharides, including but not limited to cellulosics,chitosans and alginates, all of which are abundant, natural biopolymers.All three types of materials allow for the transfer of oxygen andmoisture required to metabolize the wound healing physiology.

The natural origin of these polysaccharides varies, cellulosics arefound in plants, whereas chitosans and alginates are found in theexoskeleton or outer membrane of a variety of living organisms. Many ofthese naturally occurring polymers, in addition to being able to formlong stable chains for forming the backbone of the current invention,have other benefits that may promote further advantages for their use inenvironments of damaged tissue, hemorraghing, and/or exposed red bloodcells. For instance, chitosan also has inherent anti-microbialproperties; this is a crucial asset for materials covering open woundsbecause it eliminates the need to constantly change wound dressings inorder to disinfect the wound manually between changes. Positive chargesalong the backbone of chitosan cause it to interact electrostaticallywith negatively charged blood cells, thus creating a sticky interfacebetween a chitosan sponge and the wound. Chitosan provides hemostasisfor an extended period of time when compared against standard bandages.For example, the sponge of the current invention provides substantially30 minutes more absorption/adherence time before becoming saturated withblood cells and losing adhesion to the wound site.

The form of the natural polymers used may vary to include standardstates, derivatives and other various formulations. For example, thecellulosics may include without limitation, hydroxyetyhl cellulose,hydroxypropyl cellulose, methyl cellulose, hydroxypropyl methylcellulose, and/or hydroethyl methyl cellulose. Chitosans may includewithout limitation, the following chitosan salts: chitosan lactate,chitosan salicylate, chitosan pyrrolidone carboxylate, chitosanitaconate, chitosan niacinate, chitosan formate, chitosan acetate,chitosan gallate, chitosan glutamate, chitosan maleate, chitosanaspartate, chitosan glycolate and quaternary amine substituted chitosanand salts thereof. Alginates may include without limitation, sodiumalginate, potassium alginate, magnesium alginate, calcium alginate,and/or aluminum alginate. It is to be understood that various otherforms of any of these natural polysaccharides that provide the properfunctional capabilities may be employed without departing from the scopeand spirit of the present invention.

In alternative embodiments of this invention the polymeric component ofthe current invention is a mixture of polysaccharides. For instance, themixture may be of various different sub-classes of a single polymerclass. Alternatively, the mixture may include two or more differentclasses of polymer, for instance a cellolusic and a chitosan.

In a preferred embodiment, a matrix of the current invention is formedthrough the binding of numerous hydrophobically modified chitosancompounds, as shown in FIS. 1. These novel compounds consist of abiopolymer (e.g., chitosan) backbone that includes a hydrophilicallyreactive functional group (e.g., amino groups) that binds with thehydrophilically reactive head groups (e.g., carbonyl functional group)of an amphiphilic compound (e.g., aldehyde). The head group is furtherassociated with a hydrophobic tail group. In the current embodiment, thehydrophobic tail may be for example a hydrocarbon. Thus, a hydrophobictail is associated with the biopolymer's chitosan backbone providing thehydrophobic modification to the molecule that extends from the backboneand may interact with a surrounding environment in numerous ways, suchas through hydrophobic interaction with other tissues, cells, moleculesand/or structures. The hydrophobic interaction between the modifiedchitosan and the bi-layer of various tissues and/or cells occurs via the“insertion and anchoring” of the hydrophobic tail group of the shorthydrophobic substituent into the bi-layer membrane of the tissues orcells. The insertion process is driven by the generally understoodhydrophobic interaction and those forces that are at work which tend togroup like molecules when they exist in a heterogenous environment.Thus, the hydrophobic effect or interaction is evidenced by the tendencyof hydrophobic components to group together versus interacting orbonding with other molecules.

Typically, and for the purposes of the preferred embodiments of theinstant application, these hydrophobically modified polymers(biopolymers) are referenced as being composed of a chitosan “backbone”,“scaffold”, and/or “lattice”. Thus, the backbone of the hydrophobicallymodified biopolymer film matrix of the preferred embodiments of thecurrent invention is the biopolymer chitosan. Other biopolymers,including but not limited to the cellulosics and alginates, whichinclude similar characteristics of the chitosan backbone may be employedwith departing from the scope and spirit of the instant invention.

Chitosan is a deacetylated derivative of chitin, wherein the degree ofdeacetylation (%DA) may range from 60-100% and determines the chargedensity. Chitosan is a linear polysaccharide composed of repeatingβ-(1-4)-linked D-glucosamine monomeric units.

Chitosan structure showing three of the repeating beta-(1-4)-linkedD-glucosamine units (deacetylated).

These repeating monomeric units include a free amino group (functionalgroup) and may make molecules or compounds containing chitosan or itsderivatives readily reactive. The hydrophic modification of the chitosanbackbone is through the association of an amphiphilic compound with theamino group, such that the hydrophobic tail of the amphiphilic compoundis bound with the hydrophilic backbone structure.. As seen in FIGS. 1and 2, this hydrophobically modified chitosan backbone (hm-Chitosan) maythen be cast into a film. In the preferred embodiment of FIGS. 1 and 2,numerous hm-Chitosan backbones may fill a solution which may then becast into a film forming the novel hm-Chitosan film of the currentinvention. This film matrix is a solid-state or dried film of thehm-Chitosan.

The formation or fabrication of the novel solid-state, hm-Chitosanmatrix occurs through well known processes. The formation of the spongeof the current invention includes the formation step of freeze dryingthe solid state film of the matrix. Thus, a preferred embodiment of thecurrent invention is a freeze dried, hm-Chitosan sponge or compositionof matter which may be readily reactive with tissue, cells andadditional molecules and/or compounds. The sponge composition of matteris prepared as a readily reactive, solid-state film matrix forapplication and use in damaged tissue adhesion and red blood cellgelling for promoting hemostasis. The sponge may be a bandage orincluded as part of a bandage that may be applied to a wounded area.However, various other implementation states of the current invention asmay be contemplated by those of ordinary skill in the art are herebyassumed to fall within the scope of the current invention.

The sponge and spray solution of the current invention include at leastone polymer and a plurality of short hydrophobic substituent attachedalong the backbone of the polymer. The short hydrophobic substituentpreferably includes a hydrocarbon group having from about 8 to about 18carbon atoms attached to the backbone of the at least one polymer. Inpreferred embodiments, the hydrocarbon group comprises an alkyl orarylalkyl group. As used herein, the term “arylalkyl group” means agroup containing both aromatic and aliphatic structures. Examples ofprocedures for modifying polymers are as follows:

(1) Alginates can be hydrophobically modified by exchanging theirpositively charged counterions (e.g. Na⁺) with tertiary-butyl ammonium(TBA*) ions using a sulfonated ion exchange resin. The resultingTBA-alginate is dissolved in dimethylsulfoxide (DMSO) where reactionoccurs between alkyl (or aryl) bromides and the carboxylate groups alongthe alginate backbone.

(2) Cellulosics can be hydrophobically-modified by first treating thecellulosic material with a large excess highly basic aqueous solution(e.g. 20 wt % sodium hydroxide in water). The alkali cellulose is thenremoved from solution and vigorously mixed with an emulsifying solution(for example, oleic acid) containing the reactant, which is an alkly (oraryl) halide (e.g. dodecyl bromide).

(3) Chitosans can be hydrophobically-modified by reaction of alkyl (oraryl) aldehydes with primary amine groups along the chitosan backbone ina 50/50 (v/v) % of aqueous 0.2 M acetic acid and ethanol. Afterreaction, the resulting Schiff bases, or imine groups, are reduced tostable secondary amines by dropwise addition of the reducing agentsodium cyanoborohydride.

The degree of substitution of the hydrophobic substituent on the polymeris from about 1 to about 30 moles of the hydrophobic substituent permole of the polymer, which hydrophobic substitutions occur in up to 10%of available amines of the chitosan backbone, preferably between 1.5%and 4.5%. It is contemplated that more than one particular hydrophobicsubstituent is substituted onto the polymer, provided that the totalsubstitution level is substantially within the ranges set forth above.

The short hydrophobic substituent is an amphiphilic compound meaning itis composed of a hydrophilic Head group and a hydrophobic Tail group.The Head group binds with the polymer and positions the Tail group toextend from the backbone of the polymer scaffold. This makes thehydrophobic Tail group available for hydrophobic interactions. The Tailgroup is preferably a hydrocarbon of various forms. As used herein,hydrocarbon(s) are any organic molecule(s) or compound(s) with a“backbone” or “skeleton” consisting entirely of hydrogen and carbonatoms and which lack a functional group. Thus, these types of compoundsare hydrophobic in nature, unable to react hydrophilically, andtherefore provide an opportunity for hydrophobic interaction. Thehydrophobic interaction capability of the amphiphilic compound bound tothe chitosan backbone may provide significant advantage to the currentinvention when compared to the prior art in that the interaction of thehydrophobically modified polymer matrix, whether chitosan, cellulosic oralginate based, with the bi-layer membrane of tissue(s) and cell(s) is aself-driven, thermodynamic process requiring less energy input. Thus,regardless of any particular form of the Tail group of the shorthydrophobic substituent (amphiphilic compound), so long as it providesthe opportunity for hydrophobic interaction with the tissue(s), cell(s),or other hydrophobically active molecules and/or compounds it fallswithin the scope and spirit of the current invention.

Hydrocarbons, which are hydrophobic, may form into various types ofcompounds/molecules, such as gases (e.g. methane and propane), liquids(e.g., hexane and benzene), waxes or low melting solids (e.g., paraffinwas and naphthalene), polymers (e.g., polyethylene, polypropylene andpolystyrene), or biopolymers. Currently, hydrocarbons may be classifiedas follows:

(1) Saturated Hydrocarbons (alkanes) are composed entirely of singlebonds between the carbon and hydrogen atoms and are denoted by (assumingnon-cylcic structures) the general formula C_(n)H_(2n+2). These types ofcompounds are the most simple of the hydrocarbons and are either foundas linear or branched species of unlimited number.

(2) Unsaturated Hydrocarbons include one or more multiple bonds betweencarbon atoms of the compound, such as double bonds (alkenes-C_(n)H_(2n))or triple bonds (alkynes-C_(n)H_(2n−2)). These multiple bonds createcarbon atoms which are also commonly referred to as hydrogenated in thatthey are in need of the addition of further hydrogen atoms.

(3) Cycloalkanes consist of only carbon and hydrogen atoms are cyclic or“ring-shaped” alkane hydrocarbons denoted by the general formulaC_(n)H_(2(n−1-g)) where n=number of C atoms and g=number of rings in themolecule. Cycloalkanes are saturated because there are no multiple(double or triple) C-C bonds to hydrogenate (add more hydrogen to).

(4) Aromatic Hydrocarbons, also known as arenes, are hydrocarbons thatcontain at least one aromatic ring and may be denoted by the formulaC_(n)H_(n), wherein at a minimum n=6. Arenes (e.g., Benzene-C₆H₆) orAromatic Hydrocarbons include a molecular structure which incorporatesone or more planar sets of six carbon atoms connected by delocalizedelectrons numbering the same as if they consisted of alternating singleand double covalent bonds.

From this basic classification system there exist many derivatives andfurther types of compounds that build therefrom. For example, numerousand varied compounds include more than one aromatic ring and aregenerally referred to as polyaromatic hydrocarbons (PAH); they are alsocalled polycyclic aromatic hydrocarbons and polynuclear aromatichydrocarbons. Various alternative/derivative forms of the saturated orunsaturated cycloalkanes, and aromatic hydrocarbons as are known andcontemplated by those skilled in the art may be employed with thecurrent invention and should be read as falling within the contemplatedscope of the current invention.

Various types of other hydrophobic, organic compounds may generallyinclude hydrocarbon backbones but may also include other types of atomsand/or incorporate/bind to other compounds/molecular structures thatincorporate other types of atoms than just carbon and hydrogen. Thus,another classification system has developed by which organic compoundswith generally hydrocarbon backbones but bound with other types ofmolecules may be separated, wherein such compounds may be designatedeither aromatic or aliphatic. Thus, compounds composed mainly,substantially or at least partially, but not exclusively of carbon andhydrogen may be divided into two classes:

1. aromatic compounds, which contain benzene and other similarcompounds, and

2. aliphatic compounds (G. aleiphar, fat, oil), which do not.

In aliphatic compounds, carbon atoms can be joined together in straightchains, branched chains, or rings (in which case they are calledalicyclic). They can be joined by single bonds (alkanes), double bonds(alkenes), or triple bonds (alkynes). Besides hydrogen, other elementscan be bound to the carbon chain, the most common being oxygen,nitrogen, sulfur, and chlorine. Those of ordinary skill in the art willrecognize that other molecules may also be bound to the carbon chainsand that compounds of such heteroatomic structure are contemplated asfalling within the scope of the current invention.

The hydrophobic Tail group of the amphiphilic compound bound to thepolymer backbone of the current invention is capable of branching and/orallowing the inclusion of side chains onto its carbon backbone. This maypromote the hydrophobic interaction between the hydrophobically modifiedpolymer matrix and damaged tissue and/or cell, as discussed throughoutthe instant specification. It may be understood that the strength of thehydrophobic interaction is based upon the available amount of“hydrophobes” that may interact amongst themselves or one another. Thus,it may further promote the hydrophobic effect by increasing the amountof and/or “hydrophobic” nature of the hydrophobic Tail group that isinteracting. For instance, a hydrophobic Tail group, which in itsoriginal form may include a hydrocarbon chain, may promote an increasein its hydrophobicity (ability to hydrophobically bond and strength ofhydrophic interaction) by having a hydrophobic side chain attach to oneof the carbons of its carbon backbone. In a preferred embodiment of thecurrent invention, this may include the attachment of various polycycliccompounds, which may include for instance various steroidal compoundsand/or their derivatives such as sterol type compounds, moreparticularly cholesterol.

In alternative embodiments, the current invention contemplates the useof various molecules and/or compounds that may increase the hydrophobicinteraction allowed between the Tail group and the bi-layer membrane oftissues and cells. The side chains may be linear chains, aromatic,aliphatic, cyclic, polycyclic, or any various other types of hydrophobicside chains as contemplated by those skilled in the art. Some of thecontemplated hydrophobic side chains may include the following:

I. Linear Alkanes

Number of C atoms Formula Common name Synonyms 1 CH₄ Methane marsh gas;methyl hydride; natural gas 2 C₂H₆ Ethane dimethyl; ethyl hydride;methyl methane 3 C₃H₈ Propane dimethyl methane; propyl hydride 4 C₄H₁₀n-Butane butyl hydride; methylethyl methane 5 C₅H₁₂ n-Pentane amylhydride; Skellysolve A 6 C₆H₁₄ n-Hexane dipropyl; Gettysolve-B; hexylhydride; Skellysolve B 7 C₇H₁₆ n-Heptane dipropyl methane; Gettysolve-C;heptyl hydride; Skellysolve C 8 C₈H₁₈ n-Octane dibutyl; octyl hydride 9C₉H₂₀ n-Nonane nonyl hydride; Shellsol 140 10 C₁₀H₂₂ n-Decane decylhydride 11 C₁₁H₂₄ n-Undecane hendecane 12 C₁₂H₂₆ n-Dodecane adakane 12;bihexyl; dihexyl; duodecane 13 C₁₃H₂₈ nTridecane 14 C₁₄H₃₀ n-Tetradecane15 C₁₅H₃₂ n-Pentadecane 16 C₁₆H₃₄ n-Hexadecane cetane 17 C₁₇H₃₆n-Heptadecane 18 C₁₈H₃₈ n-Octadecane 19 C₁₉H₄₀ n-Nonadecane 20 C₂₀H₄₂n-Eicosane didecyl 21 C₂₁H₄₄ n-Heneicosane 22 C₂₂H₄₆ n-Docosane 23C₂₃H₄₈ n-Tricosane 24 C₂₄H₅₀ n-Tetracosane tetrakosane 25 C₂₅H₅₂n-Pentacosane 26 C₂₆H₅₄ n-Hexacosane cerane; hexeikosane 27 C₂₇H₅₆n-Heptacosane 28 C₂₈H₅₈ n-Octacosane 29 C₂₉H₆₀ n-Nonacosane 30 C₃₀H₆₂n-Triacontane 31 C₃₁H₆₄ n-Hentraiacontane untriacontane 32 C₃₂H₆₆n-Dotriacontane dicetyl 33 C₃₃H₆₈ n-Tritriacontane 34 C₃₄H₇₀n-Tetratriacontane 35 C₃₅H₇₂ n-Pentatriacontane 36 C₃₆H₇₄n-Hexatriacontane 37 C₃₇H₇₆ n-Heptatriacontane 38 C₃₈H₇₈n-Octatriacontane 39 C₃₉H₈₀ n-Nonatriacontane 40 C₄₀H₈₂ n-Tetracontane41 C₄₁H₈₄ n-Hentetracontane 42 C₄₂H₈₆ n-Dotetracontane 43 C₄₃H₈₈n-Tritetracontane 44 C₄₄H₉₀ n-Tetratetracontane 45 C₄₅H₉₂n-Pentatetracontane 46 C₄₆H₉₄ n-Hexatetracontane 47 C₄₇H₉₆n-Heptatetracontane 48 C₄₈H₉₈ n-Octatetracontane 49 C₄₉H₁₀₀n-Nonatetracontane 50 C₅₀H₁₀₂ n-Pentacontane 51 C₅₁H₁₀₄n-Henpentacontane 52 C₅₂H₁₀₆ n-Dopentacontane 53 C₅₃H₁₀₈n-Tripentacontane 54 C₅₄H₁₁₀ n-Tetrapentacontane 55 C₅₅H₁₁₂n-Pentapentacontane 56 C₅₆H₁₁₄ n-Hexapentacontane 57 C₅₇H₁₁₆n-Heptapentacontane 58 C₅₈H₁₁₈ n-Octapentacontane 59 C₅₉H₁₂₀n-Nonapentacontane 60 C₆₀H₁₂₂ n-Hexacontane 61 C₆₁H₁₂₄ n-Henhexacontane62 C₆₂H₁₂₆ n-Dohexacontane 63 C₆₃H₁₂₈ n-Trihexacontane 64 C₆₄H₁₃₀n-Tetrahexacontane 65 C₆₅H₁₃₂ n-Pentahexacontane 66 C₆₆H₁₃₄n-Hexahexacontane 67 C₆₇H₁₃₆ n-Heptahexacontane 68 C₆₈H₁₃₈n-Octahexacontane 69 C₆₉H₁₄₀ n-Nonahexacontane 70 C₇₀H₁₄₂ n-Heptacontane71 C₇₁H₁₄₄ n-Henheptacontane 72 C₇₂H₁₄₆ n-Doheptacontane 73 C₇₃H₁₄₈n-Triheptacontane 74 C₇₄H₁₅₀ n-Tetraheptacontane 75 C₇₅H₁₅₂n-Pentaheptacontane 76 C₇₆H₁₅₄ n-Hexaheptacontane 77 C₇₇H₁₅₆n-Heptaheptacontane 78 C₇₈H₁₅₈ n-Octaheptacontane 79 C₇₉H₁₆₀n-Nonaheptacontane 80 C₈₀H₁₆₂ n-Octacontane 81 C₈₁H₁₆₄ n-Henoctacontane82 C₈₂H₁₆₆ n-Dooctacontane 83 C₈₃H₁₆₈ n-Trioctacontane 84 C₈₄H₁₇₀n-Tetraoctacontane 85 C₈₅H₁₇₂ n-Pentaoctacontane 86 C₈₆H₁₇₄n-Hexaoctacontane 87 C₈₇H₁₇₆ n-Heptaoctacontane 88 C₈₈H₁₇₈n-Octaoctacontane 89 C₈₉H₁₈₀ n-Nonaoctacontane 90 C₉₀H₁₈₂ n-Nonacontane91 C₉₁H₁₈₄ n-Hennonacontane 92 C₉₂H₁₈₆ n-Dononacontane 93 C₉₃H₁₈₈n-Trinonacontane 94 C₉₄H₁₉₀ n-Tetranonacontane 95 C₉₅H₁₉₂n-Pentanonacontane 96 C₉₆H₁₉₄ n-Hexanonacontane 97 C₉₇H₁₉₆n-Heptanonacontane 98 C₉₈H₁₉₈ n-Octanonacontane 99 C₉₉H₂₀₀n-Nonanonacontane 100 C₁₀₀H₂₀₂ n-Hectane 101 C₁₀₁H₂₀₄ n-Henihectane 102C₁₀₂H₂₀₆ n-Dohectane 103 C₁₀₃H₂₀₈ n-Trihectane 104 C₁₀₄H₂₁₀n-Tetrahectane 105 C₁₀₅H₂₁₂ n-Pentahectane 106 C₁₀₆H₂₁₄ n-Hexahectane107 C₁₀₇H₂₁₆ n-Heptahectane 108 C₁₀₈H₂₁₈ n-Octahectane 109 C₁₀₉H₂₂₀n-Nonahectane 110 C₁₁₀H₂₂₂ n-Decahectane 111 C₁₁₁H₂₂₄ n-Undecahectane

II. Cyclic Compounds

Cyclic compounds can be categorized:

Alicyclic Compound An organic compound that is both aliphaticCycloalkane and cyclic with or without side chains Cycloalkene attached.Typically include one or more all- carbon rings (may be saturated orunsaturated), but NO aromatic character. Aromatic hydrocarbon See aboveand below Polycyclic aromatic hydrocarbon Heterocyclic compound Organiccompounds with a ring structure containing atoms in addition to carbon,such as nitrogen, oxygen, sulfur, chloride as part of the ring. May besimple aromatic rings or non-aromatic rings. Some examples are Pyridine(C5H5N), Pyrimidine (C4H4N2) and Dioxane (C4H8O2). Macrocycle See below.

III. Polycyclic Compounds—polycyclic compound is a cyclic compound withmore than one hydrocarbon loop or ring structures (Benzene rings). Theterm generally includes all polycyclic aromatic compounds, including thepolycyclic aromatic hydrocarbons, the heterocyclic aromatic compoundscontaining sulfur, nitrogen, oxygen, or another non-carbon atoms, andsubstituted derivatives of these. The following is a list of some knownpolycyclic compounds.

Example Polycyclic Compounds Sub-Types Compounds Bridged Compound-Bicyclo compound adamantane compounds which contain amantadineinterlocking rings biperiden memantine methenamine rimantadineMacrocyclic Compounds- Calixarene any molecule containing a CrownCompounds ring of seven, fifteen, or any Cyclodextrins arbitrarily largenumber of Cycloparaffins atoms Ethers, cyclic Lactams, macrocyclicMacrolides Peptides, cyclic Tetrapyrroles Trichothecenes PolycyclicHydrocarbons, Acenaphthenes Aromatic Anthracenes AzulenesBenz(a)anthracenes Benzocycloheptenes Fluorenes Indenes NaphthalenesPhenalenes Phenanthrenes Pyrenes Spiro Compounds Steroids AndrostanesBile Acids and Salts Bufanolides Cardanolides Cholanes ChoestanesCyclosteroids Estranes Gonanes Homosteroids Hydroxysteroids KetosteroidsNorsteroids Prenanes Secosteroids Spirostans Steroids, BrominatedSteroids, Chlorinated Steroids, Fluorinated Steroids, Heterocyclic

The addition of the side chains may increase the stability and strengthof the hydrophobic interaction between the Tail group and otherhydrophobically active locations, such as a hydrophobic cavity in thebi-layer membrane of various biological structures including tissue andcell membrane structures. This increase in strength and stability mayprovide further advantages in the ability of the hydrophobicallymodified polymer matrix to self-assemble, such as providing increased orstabilized rates of reaction in the formation of the network film. Theability to adjust the side chain hydrophobicity may directly impact uponthe tertiary and quaternary structure of the hydrophobically modifiedpolymer matrix either as a reactive, solid-state matrix or as aliquid-state solution.

The molecular weight of the polymers comprising the tissue sealantsponge ranges from about 50,000 to about 500,000 grams per gram mole. Itis contemplated that the molecular weight of the polymers in the spongeor solution formulations may be less than or greater than the rangeidentified without departing from the scope and spirit of the currentinvention. For instance, the molecular weight of the polymers comprisingthe spray ranges from about 10,000 to about 200,000 grams per gram mole.As used herein, the term “molecular weight” means weight averagemolecular weight. In preferred examples, average molecular weight ofpolymers is determined by low angle laser light scattering (LLS) andSize Exclusion Chromatography (SEC). In performing low angle LLS, adilute solution of the polymer, typically 2% or less, is placed in thepath of a monochromatic laser. Light scattered from the sample hits thedetector, which is positioned at a low angle relative to the lasersource. Fluctuation in scattered light over time is correlated with theaverage molecular weight of the polymer in solution. In performing SECmeasurements, again a dilute solution of polymer, typically 2% or less,is injected into a packed column. The polymer is separated based on thesize of the dissolved polymer molecules and compared with a series ofstandards to derive the molecular weight.

The hydrophobically modified polymer spray solution is mixed with avariety of water-soluble reagents which results in faster and moreefficient healing of the wound in alternative embodiments of the currentinvention. A first class of reagents that is mixed with thehydrophobically modified polymer is comprised of those reagents thatcontribute to the hemostatic integrity of the clot such as for examplehuman thrombin, bovine thrombin, recombinant thrombin, and any of thesethrombins in combination with human fibrinogen. Other examples of thefirst class of reagents include fibrinogen and Factor XIII. A secondclass of reagents that is mixed with the hydrophobically modifiedpolymer is comprised of those reagents that prevent microbial infectionsuch as norfloxacin, silver, ampicillin and penicillin. Reagents fromboth classes, e.g. recombinant thrombin and norfloxacin, or reagentsfrom the same class, e.g. recombinant thrombin and fibronectin, may bemixed with the polymer. Various other reagents, catalysts, excipients,transporters, and/or penetrating agents as are known in the art may beemployed by the current invention.

In another preferred embodiment of the current invention, the solutionof the hydrophobically modified polymer matrix is formulated into anovel adhesive foam. Therefore, the current invention contemplates amethod of preparing a foam of the hydrophobically modified polymermatrix. The foam formulation and development techniques employed mayvary, including development by standard mechanical agitation means,freeze-dried foam, and various other techniques and formulations as maybe contemplated by those of ordinary skill in the art. For instance, thefoam may be produced by beating or otherwise agitating thepolysaccharide polymer, including the plurality of short hydrophobicsubstituents, until it foams. It is also contemplated that depending onthe polymer being used to prepare the foam, the foaming process takesplace in an acidic solution or aqueous base.

It is contemplated that the foaming process may include the introductionof various other materials, such as various gases, into the solutionthat is being foamed. Different means of mixing the various other gasesinto the solution to provide a dispersion throughout the solution may beemployed. Various foaming agents, modifiers, plasticizers and/orstabilizers may also be employed by the current invention to assist infoaming the solution. For instance, various ionic or non-ionicsurfactants, cross-linkers or coagulant stabilizers may be used. It isalso further contemplated that the various physical dimensions of andwithin the foam may be modified and/or controlled by various means ascontemplated by those skilled in the art and such means may be employedwithout departing from the scope and spirit of the current invention.

The various forms of the novel composition of matter provided by thecurrent invention may be used separately and independently. It is alsocontemplated that these various forms, whether sponge, solution and/orfoam, may be employed in combination to provide their beneficial effect.It is also contemplated that one or more the different forms may bemixed and/or blended together for use as a combination product. It iscontemplated that the different forms may include similar or differentformulations of the novel matrix of the current invention. Theinteraction of the different forms may be promoted or affected throughthe use of various different agents as may be contemplated by those ofordinary skill in the art.

In FIG. 4 a method 400 of using a hydrophobically modified biopolymermatrix is provided. The method includes a step 410 of administering apolymer attached with a plurality of short hydrophobic substituent to awounded tissue or a red blood cell, wherein the attached plurality ofshort hydrophobic substituent is capable, through hydrophobicinteraction, of adhering with the wounded tissue or red blood cell.Administration may preferably occur through directly contacting thepolymer matrix with the wounded tissue and/or cells and the applicationof pressure. The pressure is sustained thereby maintaining contactbetween the polymer matrix and the wounded tissue and/or cells. It iscontemplated that the hydrophobically modified biopolymer matrix isformulated as a solid state sponge that is administered to awound/damaged tissue by pressing the sponge firmly against a wound area.It is contemplated that the length of time for which pressure is appliedmay vary, but perhaps most preferably pressure is applied for about 5 toabout 10 seconds. The administration of the sponge to the wounded areaallows for the interaction between the hydrophobically modified polymermatrix and the damaged tissue. By contacting of the sponge to thedamaged tissue the hydrocarbon chains of the plurality of shorthydrophobic substituent are able to anchor into the tissue throughhydrophobic interaction.

In an alternative embodiment, the hydrophobically modified biopolymermatrix is formulated as an aqueous solution of about 1 to about 2.5% byweight of the polymer. The solution may be administered by spraying itonto a wound area whereby the matrix of the current invention is allowedto hydrophobically interact with the red blood cells and damaged tissueto promote the prevention of blood loss. In either embodiment, sponge orsolution, the current invention provides for increased tissue andcellular adhesion which promotes the hemostatic progression in a woundedarea.

A method of manufacturing the sponge and/or solution of the currentinvention is contemplated as an independent method or as a part of themethod of using the hydrophobically modified biopolymer matrix, ineither the sponge or solution formulation, prior to the administrationstep. It is contemplated that the sponge is manufactured by dehydratingthe hydrophobically modified polymer dissolved in aqueous solution. In afirst step, an aqueous liquid solution containing between about 1 andabout 2.5% by weight of the hydrophobically modified polymer is castinto a container having the desired dimensions of the sponge. In anexample, the container is substantially sealed and frozen in a solutionof liquid nitrogen and left in the liquid nitrogen solution for about 20to about 30 seconds. The container is then opened and placed into avacuum chamber attached to a freeze drying system which dehydrates thepolysaccharide over a period of about 24 hours.

In another example, the container is cast as described above and isplaced inside a vacuum oven at about 60° C. for a period of about 48hours to produce a thin, flexible absorbent film material that is ableto be used as a bandage wrap for wounds with extraordinary geometrieswhich are difficult to treat with freeze-dried sponges.

A system of the current invention includes a container that is used tostore the solution form of the hydrophobically modified matrix. Thecontainer is operationally connected to an ejection mechanism or spraymechanism which is capable of ejecting the solution from the containerby means of applying a mechanical pressure to the fluid. The ejectionmechanism may be a manual spray mechanism or may provide itsfunctionality through the use of engaging any type of actuator(mechanical, electrical, magnetic, and the like).

In an alternative embodiment of the system, there may be two or morecontainers wherein a first container stores the solution and a secondcontainer stores at least one secondary component like a reagent such asthrombin or norfloxacin, as described above. It is contemplated that thesecondary component may be mixed with the hydrophobically modifiedpolymer solution in the same reservoir, or is simultaneously dispensedfrom a separate chamber. It is further contemplated that the solutionand the secondary component may be mixed together prior to ejection. Themeans used to blend the solution and secondary components may be anysuch means as contemplated by those of ordinary skill in the art.

The following methods and results provide an exemplary formulation ofthe matrix of the current invention and its efficacy in promotinghemostasis. It is understood that alternative formulations, such asthose explicitly described in the instant specification as well as thosethat are contemplated by those of ordinary skill in the art, may be usedand achieve similar results.

Chitosan was hydrophobically modified in order to increase its adhesionto tissue at the site of injury and to improve its hemostaticproperties. Hydrophobes were attached along the backbone of the chitosanpolymer. Based on previous work done by Lee et al, the workinghypothesis for the present study is that hydrophobes will insertthemselves into the bilayers of cells, thus providing chitosan with anadded functionality in treating acute wounds. (See J. H. Lee, J. P.Gustin, T. Chen, G. F. Payne and S. R. Raghavan, “Vesicle-biopolymergels: Networks of surfactant vesicles connected by associatingbiopolymers.” Langmuir. (2005), which is herein incorporated byreference in its entirety). If applied to a hemostatic bandage, thisproperty could overcome the primary problem with the HemCon bandagewhile maintaining the advantages of using chitosan as the base polymer.

Due to these advanced properties of the hm-chitosan, the biopolymer is aviable option for usage as a strongly adhesive hemostatic bandage aswell as a flowable spray or surgical sealant to stop minor bleeding andto seal tissues in surgical applications. In this study, the tissueadhesion relative to the level of hydrophobic modification was observedas well as the use of hm-chitosan to gel blood.

Materials and Methods; Hydrophobic Modification of Chitosan;Synthesizing hm-Chitosan Using Dodecaldehyde.

Two grams of chitosan was dissolved in 100 mL of 0.2 M acetic acid bystirring for 30 minutes in a beaker covered with aluminum foil. Thesolution was filtered using a vacuum filter. Once the chitosan solutionwas poured from the flask into a 600 mL beaker, 100 mL of ethanol wasadded to the flask gradually and swirled around to remove the remainingchitosan on the sides of the flask. The ethanol and remaining chitosanwas poured into the beaker with the rest of the chitosan. In a separatebeaker, 20 mL of ethanol was added to dodecaldehyde (27.9 μL for the 1%,69.7 μL for the 2.5%, 97.8 μL for the 3.5%, and 167 μL for the 6%modification), which was then slowly poured into the chitosan solution.Sodium cyanoborohydride, 0.78 g, was dissolved in 10 mL of ethanol andadded to the chitosan solution. The sodium cyanoborohydride and ethanolsolution was added twice more at 2 hour intervals. The mixture wasstirred for 24 hours and the hm-chitosan was then precipitated from thesolution by adding 0.2 M sodium hydroxide dropwise.

Synthesizing hm-Chitosan Using 4-octadecyloxybenzaldehyde (2.5%Modification).

The same procedure to modify the chitosan using the dodecaldehyde isused, except 0.1178 g of 4-octadecyloxybenzaldehyde was added instead ofthe dodecaldehyde. In this study, only a 2.5% modification was madeusing the 4-octadecyloxybenzaldehyde.

Purification of hm-Chitosan Solutions.

After precipitating the hm-chitosan, the solution was poured intocentrifuge tubes, equalized in weight by adding ethanol, and centrifugedat 3,000 rpm for 10 minutes. The supernatant was removed and 20-25 mL ofethanol was added to each tube, stirred using a vortex, and centrifugedagain. This process was repeated for a total of three times usingethanol and three additional times using deionized water.

Tissue Adhesion Experiments; Preparation of Films.

After the purification process, the hm-chitosan was removed from thecentrifuge tubes into a beaker and an arbitrary amount of 0.2 M aceticacid so that the hm-chitosan was completely submerged. The solution wasstirred for 2 hours and then poured into aluminum foil. It was vacuumdried to remove all of the water. Once dry, the hm-chitosan sheets wereremoved from the foil and dissolved in 0.2 M acetic acid to form a 2 wt% solution. Then 11 g of the hm-chitosan (or chitosan) was dropped intoa 2.25″ diameter plastic petri dish and left overnight to dry under thehood.

Bovine Tissue Adhesion Tests—By Mass.

The dried films were removed from the petri dishes very carefully toprevent tearing the edges. Each film was cut in half and each pieceweighed. All of the films were then further cut and reweighed until allof them were approximately the same weight. The films were then placedon bovine muscle tissue and pressed down gently so that all of the film,except for the raised edges, was in contact with the tissue. For tenminutes, the films were allowed to adhere to the tissue. Then the bovinemuscle tissue was inverted and weights were hung sequentially to theedge of the film until it was fully removed from the tissue.

Bovine Tissue Adhesion Tests—By Area.

Once the films were removed from the petri dishes, two rectanglesmeasuring 1.5″ by 0.75″ each were cut from each film. With the filmarranged vertically, a line was measured and drawn 0.5″ from the bottomof the film using a thin permanent marker. The films were then placed onbovine muscle tissue so that the line was on the edge of the tissue withthe majority of the film adhered to the tissue and the 0.5″ by 0.75″rectangle hanging off the side. After being pressed gently into thetissue, the films were left to adhere to the tissue for ten minutes. Thebovine muscle tissue was then held vertically so the films wereperpendicular to the floor with the unattached portion of the film onthe bottom. Weights were hung sequentially to the films until they werecompletely peeled from the tissue.

Texture Analyzer.

A TA.XT2i Texture Analyzer with a 5 kg load cell was used to performthis set of experiments. The instrument was set to the compression testmode. Small viles were filled approximately half full with 2 wt %solutions of hm-chitosan of varying levels of modification as well asthe unmodified chitosan. A ½″ diameter cylindrical probe was attachedand aligned with the vile of solution. The settings were applied with apre-test speed of 3 mm/s, a test speed of 2 mm/s, and a post test speedof 1 mm/s. The target mode was distance and the distance the probetraveled after touching the surface of the solution was set to 2 mm. Asthe probe was lowered into the vile, the force on the probe when it camein contact with the solution triggered the instrument to record thedata. The resulting graphs produced were then used to calculate theadhesion of each solution.

Blood Gelling Experiments; Initial Blood Gelling Tests.

When the human blood was drawn, it was put into test tubes whoseinterior walls were lined with heparin. A pipette was then used tomeasure out 1 mL of blood, which was relocated to another test tube.Another pipette was used to add 1 mL of the chitosan (or hm-chitosan) tothe blood. During the experiments, the 1%, 2.5%, and 3.5% modifiedhm-chitosan (modified using dodecaldehyde) was used as well as 2.5%modified hm-chitosan that was modified with the4-octadecyloxybenzaldehyde. The time that it took for the mixture to gelwas observed and recorded.

Rheology of Blood and Chitosan Solution.

A AR2000 advanced rheometer with a cone and plate geometry was used tomeasure the dynamic viscoelastic properties for these experiments. Thecone had a 40 mm diameter with a 2 degree angle. In order to ensure thatall the measurements were within the linear viscoelastic regions, firststress amplitude sweeps were performed. After the human blood was drawninto the test tubes with the heparin, a pipette was used to add 1 mL ofblood onto the plate of the rheometer. Then 1 mL of the chitosan (orhm-chitosan) solution was added to the blood on the plate. Once thesolutions were combines, the parameters for the rheometer were set upand the run was started. The cone lowered into contact with the solutionand a sinusoidal strain was subjected to the subject with increasingfrequency of oscillations. The elastic and viscous moduli were obtainedover the frequency range of 0.01 to 10 Hz. Dynamic rheology experimentswere performed using unmodified chitosan, 2.5% and 6% modifiedhm-chitosan (modified using dodecaldehyde), and 2.5% modifiedhm-chitosan modified using 4-octadecyloxybenzaldehyde.

Results and Discussion; Tissue Adhesion Experiments; Texture Analyzer.

In comparison of the varying hm-chitosan solutions, an obviousrelationship was not observed in the relative adhesion calculationsfound using the texture analyzer. As shown in Table 1 below, the 1% and3.5% modified hm-chitosan had the highest level of adhesion while theunmodified chitosan was the least adhesive.

TABLE 1 Adhesion of hm-Chitosan Solutions-Texture Analyzer Level ofModification 0% 1% 2.5% 3.5% Adhesion (g · sec) 3.126 3.409 3.200 3.404

The difference between the highest level of adhesion (1% modification)and the lowest (0% modification) was 0.278g.sec. The adhesioncalculations found does not show a trend with the increasing level ofmodification with the 2.5% modified hm-chitosan being lower than the 1%and 3.5%; however, with the unmodified chitosan having the lowestadhesion, the data indicates that the hydrophobic modification of thechitosan does increase the adhesion of the solution.

Bovine Muscle Tissue; Standardized Film Weight.

The experiments in which the chitosan and hm-chitosan films were adheredto bovine muscle tissue and removed by sequentially hanging weights fromthe inverted tissue were repeated several times. As shown in FIG. 5, theaverage force to needed to remove the films increased nearly linearlywith the increasing level of hydrophobic modification. The standarddeviation of force for each level of modification is fairly low with ahigh of 0.056 N. This precision in data significantly contributes to thecredibility of the relationship between the adhesion and modificationlevel.

In unmodified chitosan, the high adhesion to tissue is due to anelectrostatic attraction between cationic chitosan and anionic cellsurfaces. As previously mentioned, it is hypothesized that the increasein adhesion with increasing hydrophobic modification is due to theanchoring of the hydrophobes on the backbone of the chitosan into thebilayer of the tissue cells. In this study, the highest level ofmodification tested is 6% modified using dodecaldehyde, and thishm-chitosan produced the greatest adhesion to the bovine muscle tissue.Due to the hydrophobic nature of the modification, a percentage ofmodification higher than 6% would be difficult to dissolve into solutionwhich would make it unusable in this study.

Though the results of the experiments provide a nearly linearrelationship between the adhesion and the level of hydrophobicmodification, the actual measurements of the force necessary to removethe films is negligible and the main focus is on the relationship. Inthis adhesion experiment, the standardization was administered byequalizing the weights of the films and by adhering all levels ofmodification to the same piece of bovine muscle tissue. With this methodof standardization, the films were of varying surface area and thicknesswhich could have had an affect on the adhesion.

Standardized Area of Contact Between Film and Tissue.

To vary the method of standardization of the films, additional adhesionexperiments were performed using films with the same surface area incontact with the bovine muscle tissue. The films measured 0.75″ by 1.5″with the portion in contact with the tissue measuring 0.75″ by 1.00″.

TABLE 2 Weight required to remove the films of varying levels ofhydrophobic modification 0% 1% 2.5% 3.5% 6% 2.5% (C18) Trial 1  78.6451g  50.6870 g 175.8375 g 104.4592 g  82.0132 g 249.0300 g Trial 2104.2906 g 112.8136 g 129.0033 g 144.4069 g 118.2246 g 171.9973 g

In addition to the five levels of modification of the hm-chitosanmodified using dodecaldehyde that were used in the previous bovinemuscle tissue adhesion experiments, a film composed of 2.5% modifiedsolution of hm-chitosan that was modified using4-octadecyloxybenzaldehyde was also used. As shown in Table 2, the 2.5%4-octadecyloxybenzaldehyde modified hm-chitosan films required thegreatest amount of force for removal which was notably higher than theamount required for the second most adhesive film. As the firstexperiment to test the adhesion of the 4-octadecyloxybenzaldehydemodified hm-chitosan film, the results suggest that this modificationcauses an even higher increase in adhesive properties.

Shown above are the chemical structures and names of the two aldehydesused for the modification of the chitosan: a) dodecaldehyde and b)4-octadecyloxybenzaldehyde. The dodecaldehyde has a 12 carbon chainwhile the 4-octadecyloxybenzaldehyde has an 18 carbon chain connected toan oxygen atom that is bonded to a benzene ring. Whether or not theincreased adhesion is due to the increase in carbon chain length or inthe addition of the benzene ring is unknown, and this is a topic ofinterest for future experiments in this study.

This series of experiments differs significantly from the other bovinemuscle tissue adhesion experiment in terms of standardization inaddition to the forces involved. With the inversion of the tissue in theother set of experiments, the films were pulled at a 90 degree angle. Inthese experiments, the tissue was held vertically and the films werepulled along the direction of the alignment of the tissue. Due to thissetup, the force needed to remove the films was higher since there was ashear force involved in the removal and the experiment was actually atype of static load shear holding test.

Since the surface area of the films was equalized in these experiments,the weight and thickness of the films varied. Similarly to the otheradhesion experiments, the same piece of bovine muscle tissue was usedfor each trial in order to reduce variation due to the properties of thetissue.

Blood Gelling Experiments; Initial Blood Gelling—Qualitative Data.

In the blood gelling experiments, the blood that was drawn was mixedwith heparin which is an anticoagulant. The purpose of adding theheparin was to ensure that the change in blood viscosity and the bloodgellation was due to the addition of the hm-chitosan solutions and not aresult of the natural blood coagulation process. In the initialexperiments, it was observed that the addition of unmodified chitosan toblood did not have a visually apparent increase in viscosity. Themixture was very fluidic and immediately flowed when the test tube wasinverted. The blood, when mixed with 1%, 2.5%, and 3.5% modifiedhm-chitosan, became exceptionally viscous, though never gelledcompletely. A gel is characterized as a solution of infinite viscosity,and does not flow even when inverted in a test tube. In this experiment,the 1%, 2.5%, and 3.5% modified hm-chitosan, when mixed with blood,became so viscous that the solutions could remain intact for severalseconds when the test tube was inverted; however, after a few seconds,the solutions slowly flowed down the side.

The 2.5% hm-chitosan solution that was modified using the4-octadecyloxybenzaldehyde completely gelled the blood almostinstantaneously (less than 30 seconds) after being added. FIG. 6 shows aphoto of the 2.5% modified hm-chitosan, modified using4-octadecyloxybenzaldehyde, mixed with the blood in a test tube and thenanother photo of the test tube completely inverted. The gelled solutionremains in the bottom of the inverted test tube. The instantaneousgellation of the blood by the 2.5% modified hm-chitosan, modified using4-octadecyloxybenzaldehyde, is a significant characteristic and haspromising future applications as a medical spray or surgical sealant.

Rheology of Blood and hm-Chitosan; Frequency Sweep.

The qualitative observations obtained in the initial blood gellingexperiments were further quantified using an advanced rheometer. Dynamicrheology experiments ultimately yield plots of G′ (elastic modulus) andG″ (viscous modulus) as functions of frequency which are collectivelycalled the frequency spectrum of the material. For our purposes ofquantitatively defining the solutions of chitosan and blood as viscousor elastic, the graphs shown in FIG. 7, identify each solution as liquidor gel (solid) form. For the unmodified chitosan and the 2.5% modifiedhm-chitosan modified using dodecaldehyde, the results show that theviscous modulus is higher than the elastic modulus. In general terms,this data indicates that the solutions have stronger viscous propertiesthan elastic and remain in liquid form. The graph of the 2.5% modifiedhm-chitosan (dodecaldehyde) seems to show some visco-elastic propertieswith the intersection of the elastic and viscous modulous; however, forour purposes the viscous modulus is higher than the elastic modulus overmost of the frequency range and only shows elastic behavior at highfrequencies.

Both the graphs of the 6% modified hm-chitosan (dodecaldehyde) and the2.5% modified hm-chitosan (4-octadecyloxybenzaldehyde) quantifies thesolutions as gels since the elastic modulus remains above the viscousmodulus throughout the frequency range. With the dodecaldehyde 6%modified hm-chitosan and blood solution, the moduli are closer togetherthan the 4-octadecyloxybenzaldehyde 2.5% modified hm-chitosan and bloodsolution. This suggests that the elastic properties of the4-octadecyloxybenzaldehyde 2.5% modified hm-chitosan and blood solutionare more dominant than with the dodecaldehyde 6% modified hm-chitosan.

Dynamic Rheology—Time to Gellation.

The previous frequency sweep graphs quantitatively show that blood, whenmixed with two of the hm-chitosan solutions, form gels; however, theprimary factor for these solutions applicability as liquid medicalsprays or surgical sealants lies within the time of gellation after theblood and hm-chitosan solutions mix. As shown in FIG. 8, the hm-chitosanand blood solutions experience negligible variation with time after theinitial mixture. For both the blood and unmodified chitosan and theblood and 2.5% modified hm-chitosan (dodecaldehyde) solutions thatremained in liquid form, the elastic modulus and viscous modulus remainrelatively constant over time. One unexplainable anomaly in the data isshown in the graph of the unmodified chitosan and blood solution withthe initial nearly linear decrease in moduli. This is believed to be aresult of an instrumental malfunction rather than a representation ofthe visco-elastic response of the solution; however, even with theinitial decrease, the moduli quickly level out and remains constant withtime.

In this experiment, the blood and hm-chitosan solutions are mixedtogether and the cone of the advanced rheometer immediately lowers intothe solution and begins the oscillations of low frequency. As shownabove in the graphs of the 6% modified hm-chitosan (dodecaldehyde) andthe 2.5% modified hm-chitosan (4-octadecyloxybenzaldehyde) and bloodsolutions, the elastic modulus is higher than the viscous modulus forthe first measurement which is determined only seconds after thesolutions are mixed. This data corresponds with the initial bloodgelling experiment results in showing the gellation time of only a fewseconds. With nearly instantaneous gellation when it comes in contactwith blood, the application of hm-chitosan liquid solutions couldpotentially mean a significant advancement in surgical sealants andsprays.

Conclusion.

The motivation behind this study was to develop a new material withadvanced properties for use as a hemostatic bandage for acute wounds andas a flowable spray or surgical sealant to stop minor bleeding and toseal tissues in surgical procedures. With our hydrophobic modificationof chitosan using dodecaldehyde and 4-octadecyloxybenzaldehyde, theexperiments of this study focused on the impact of the modification ontissue adhesion in addition to the polymers interaction with blood. Inthe tissue adhesion experiments, it was concluded that tissueadhesiveness increases proportionally with the level of hydrophobicmodification on the chitosan backbone. The experiments on theinteraction of the modified chitosan with blood revealed that the 6%dodecaldehyde modified hm-chitosan and the 4-octadecyloxybenzaldehyde2.5% modified hm-chitosan instantly forms a gel when mixed with blood.With these results showing the impact of the hydrophobic modification ofchitosan in terms of tissue adhesion and interaction with blood,hm-chitosan has advanced qualities which give it a promising future as ahemostatic bandage and surgical sealant.

It is believed that the present invention and many of its attendantadvantages will be understood by the forgoing description. It is alsobelieved that it will be apparent that various changes may be made inthe form, construction and arrangement of the components thereof withoutdeparting from the scope and spirit of the invention or withoutsacrificing all of its material advantages. The form herein beforedescribed being merely an explanatory embodiment thereof. It is theintention of the following claims to encompass and include such changes.

What is claimed is:
 1. A method, comprising: applying a hydrophobicallymodified biopolymer solution to a wound, wherein one or more hydrophobicmoieties is covalently attached to a biopolymer backbone and wherein thesolution creates an artificial clot when exposed to blood.
 2. The methodof claim 1, wherein the biopolymer is selected from the group consistingof chitosans, alginates, and cellulosics.
 3. The method of claim 1,wherein the hydrophobic moieties comprise 8 to 18 hydro-carbon residues.4. The method of claim 1, wherein the hydrophobically modifiedbiopolymer solution has a concentration of about 1% to about 2.5% byweight relative to the total weight of the solution of the biopolymer.5. The method of claim 2, wherein the hydrophobic moieties arecovalently attached to as many as 10% of available amines of chitosan.6. The method of claim 1, wherein the hydrophobically modifiedbiopolymer solution is applied as one of an adhesive foam, flowablespray, or surgical sealant.
 7. The method of claim 1, wherein thebiopolymer is a chitosan salt.
 8. The method of claim 7, wherein thechitosan salt is selected from the group consisting of chitosan iodide,chitosan citrate, chitosan bromide, chitosan lactate, chitosansalicylate, chitosan pyrrolidone carboxylate, chitosan itaconate,chitosan niacinate, chitosan forrnate, chitosan gallate, chitosanglutamate, chitosan maleate, chitosan aspartate, and chitosan glycolate.9. The method of claim 1, wherein the solution self-assembles to createthe artificial clot.
 10. The method of claim 2, wherein the chitosansolution provides for strong tissue adhesion and cellular adhesion tocreate the artificial clot.
 11. The method of claim 2, wherein thesolution readily binds to negatively charged surfaces.
 12. A method,comprising: applying a hydrophobically modified biopolymer solution to awound, wherein one or more hydrophobic moieties is covalently attachedto a biopolymer backbone and wherein the solution creates an artificialclot when exposed to blood.; and wherein said solution is packaged in acontainer for delivery to the wound.
 13. The method of claim 12, whereinthe biopolymer is selected from the group consisting of chitosans,alginates, and cellulosics.
 14. The method of claim 12, wherein thehydrophobic moieties comprise 8 to 18 hydro-carbon residues.
 15. Themethod of claim 12, wherein the hydrophobically modified biopolymersolution has a concentration of about 1% to about 2.5% by weightrelative to the total weight of the solution of the biopolymer.
 16. Themethod of claim 13, wherein the hydrophobic moieties are covalentlyattached to as many as 10% of available amines of chitosan.
 17. Themethod of claim 12, wherein the hydrophobically modified biopolymersolution is applied as one of an adhesive foam, flowable spray, orsurgical sealant.
 18. The method of claim 12, wherein the biopolymer isa chitosan salt.
 19. The method of claim 12, wherein the solutionself-assembles to create the artificial clot.
 20. The method of claim13, wherein the solution readily binds to negatively charged surfaces.